Different approaches to identify markers for meat quality and production traits in pigs: analysis of the transcriptome in post mortem skeletal muscle and investigation of candidate genes

Due to the growing attention of consumers towards their food, improvement of quality of animal products has become one of the main focus of research. To this aim, the application of modern molecular genetics approaches has been proved extremely useful and effective. This innovative drive includes all livestock species productions, including pork. The Italian pig breeding industry is unique because needs heavy pigs slaughtered at about 160 kg for the production of high quality processed products. For this reason, it requires precise meat quality and carcass characteristics. Two aspects have been considered in this thesis: the application of the transcriptome analysis in post mortem pig muscles as a possible method to evaluate meat quality parameters related to the pre mortem status of the animals, including health, nutrition, welfare, and with potential applications for product traceability (chapters 3 and 4); the study of candidate genes for obesity related traits in order to identify markers associated with fatness in pigs that could be applied to improve carcass quality (chapters 5, 6, and 7). Chapter three addresses the first issue from a methodological point of view. When we considered this issue, it was not obvious that post mortem skeletal muscle could be useful for transcriptomic analysis. Therefore we demonstrated that the quality of RNA extracted from skeletal muscle of pigs sampled at different post mortem intervals (20 minutes, 2 hours, 6 hours, and 24 hours) is good for downstream applications. Degradation occurred starting from 48 h post mortem even if at this time it is still possible to use some RNA products. In the fourth chapter, in order to demonstrate the potential use of RNA obtained up to 24 hours post mortem, we present the results of RNA analysis with the Affymetrix microarray platform that made it possible to assess the level of expression of more of 24000 mRNAs. We did not identify any significant differences between the different post mortem times suggesting that this technique could be applied to retrieve information coming from the transcriptome of skeletal muscle samples not collected just after slaughtering. This study represents the first contribution of this kind applied to pork. In the fifth chapter, we investigated as candidate for fat deposition the TBC1D1 [TBC1 (tre-2/USP6, BUB2, cdc16) gene. This gene is involved in mechanisms regulating energy homeostasis in skeletal muscle and is associated with predisposition to obesity in humans. By resequencing a fragment of the TBC1D1 gene we identified three synonymous mutations localized in exon 2 (g.40A>G, g.151C>T, and g.172T>C) and 2 polymorphisms localized in intron 2 (g.219G>A and g.252G>A). One of these polymorphisms (g.219G>A) was genotyped by high resolution melting (HRM) analysis and PCR-RFLP. Moreover, this gene sequence was mapped by radiation hybrid analysis on porcine chromosome 8. The association study was conducted in 756 performance tested pigs of Italian Large White and Italian Duroc breeds. Significant results were obtained for lean meat content, back fat thickness, visible intermuscular fat and ham weight. In chapter six, a second candidate gene (tribbles homolog 3, TRIB3) is analyzed in a study of association with carcass and meat quality traits. The TRIB3 gene is involved in energy metabolism of skeletal muscle and plays a role as suppressor of adipocyte differentiation. We identified two polymorphisms in the first coding exon of the porcine TRIB3 gene, one is a synonymous SNP (c.132T> C), a second is a missense mutation (c.146C> T, p.P49L). The two polymorphisms appear to be in complete linkage disequilibrium between and within breeds. The in silico analysis of the p.P49L substitution suggests that it might have a functional effect. The association study in about 650 pigs indicates that this marker is associated with back fat thickness in Italian Large White and Italian Duroc breeds in two different experimental designs. This polymorphisms is also associated with lactate content of muscle semimembranosus in Italian Large White pigs. Expression analysis indicated that this gene is transcribed in skeletal muscle and adipose tissue as well as in other tissues. In the seventh chapter, we reported the genotyping results for of 677 SNPs in extreme divergent groups of pigs chosen according to the extreme estimated breeding values for back fat thickness. SNPs were identified by resequencing, literature mining and in silico database mining. analysis, data reported in the literature of 60 candidates genes for obesity. Genotyping was carried out using the GoldenGate (Illumina) platform. Of the analyzed SNPs more that 300 were polymorphic in the genotyped population and had minor allele frequency (MAF) >0.05. Of these SNPs, 65 were associated (P<0.10) with back fat thickness. One of the most significant gene marker was the same TBC1D1 SNPs reported in chapter 5, confirming the role of this gene in fat deposition in pig. These results could be important to better define the pig as a model for human obesity other than for marker assisted selection to improve carcass characteristics.

Bio-physical interactions and feedbacks in a global climate model

This PhD thesis addresses the topic of large-scale interactions between climate and marine biogeochemistry. To this end, centennial simulations are performed under present and projected future climate conditions with a coupled ocean-atmosphere model containing a complex marine biogeochemistry model. The role of marine biogeochemistry in the climate system is first investigated. Phytoplankton solar radiation absorption in the upper ocean enhances sea surface temperatures and upper ocean stratification. The associated increase in ocean latent heat losses raises atmospheric temperatures and water vapor. Atmospheric circulation is modified at tropical and extratropical latitudes with impacts on precipitation, incoming solar radiation, and ocean circulation which cause upper-ocean heat content to decrease at tropical latitudes and to increase at middle latitudes. Marine biogeochemistry is tightly related to physical climate variability, which may vary in response to internal natural dynamics or to external forcing such as anthropogenic carbon emissions. Wind changes associated with the North Atlantic Oscillation (NAO), the dominant mode of climate variability in the North Atlantic, affect ocean properties by means of momentum, heat, and freshwater fluxes. Changes in upper ocean temperature and mixing impact the spatial structure and seasonality of North Atlantic phytoplankton through light and nutrient limitations. These changes affect the capability of the North Atlantic Ocean of absorbing atmospheric CO2 and of fixing it inside sinking particulate organic matter. Low-frequency NAO phases determine a delayed response of ocean circulation, temperature and salinity, which in turn affects stratification and marine biogeochemistry. In 20th and 21st century simulations natural wind fluctuations in the North Pacific, related to the two dominant modes of atmospheric variability, affect the spatial structure and the magnitude of the phytoplankton spring bloom through changes in upper-ocean temperature and mixing. The impacts of human-induced emissions in the 21st century are generally larger than natural climate fluctuations, with the phytoplankton spring bloom starting one month earlier than in the 20th century and with ~50% lower magnitude. This PhD thesis advances the knowledge of bio-physical interactions within the global climate, highlighting the intrinsic coupling between physical climate and biosphere, and providing a framework on which future studies of Earth System change can be built on.

Nanocrystalline Silicon Based Films for Renewable Energy Applications

The present thesis is focused on the study of innovative Si-based materials for third generation photovoltaics. In particular, silicon oxi-nitride (SiOxNy) thin films and multilayer of Silicon Rich Carbide (SRC)/Si have been characterized in view of their application in photovoltaics. SiOxNy is a promising material for applications in thin-film solar cells as well as for wafer based silicon solar cells, like silicon heterojunction solar cells. However, many issues relevant to the material properties have not been studied yet, such as the role of the deposition condition and precursor gas concentrations on the optical and electronic properties of the films, the composition and structure of the nanocrystals. The results presented in the thesis aim to clarify the effects of annealing and oxygen incorporation within nc-SiOxNy films on its properties in view of the photovoltaic applications. Silicon nano-crystals (Si NCs) embedded in a dielectric matrix were proposed as absorbers in all-Si multi-junction solar cells due to the quantum confinement capability of Si NCs, that allows a better match to the solar spectrum thanks to the size induced tunability of the band gap. Despite the efficient solar radiation absorption capability of this structure, its charge collection and transport properties has still to be fully demonstrated. The results presented in the thesis aim to the understanding of the transport mechanisms at macroscopic and microscopic scale. Experimental results on SiOxNy thin films and SRC/Si multilayers have been obtained at macroscopical and microscopical level using different characterizations techniques, such as Atomic Force Microscopy, Reflection and Transmission measurements, High Resolution Transmission Electron Microscopy, Energy-Dispersive X-ray spectroscopy and Fourier Transform Infrared Spectroscopy. The deep knowledge and improved understanding of the basic physical properties of these quite complex, multi-phase and multi-component systems, made by nanocrystals and amorphous phases, will contribute to improve the efficiency of Si based solar cells.

Light energy management in peach: utilization, photoprotection , photodamage and recovery. Maximizing light absorption in orchard is not always the best solution

The relation between the intercepted light and orchard productivity was considered linear, although this dependence seems to be more subordinate to planting system rather than light intensity. At whole plant level not always the increase of irradiance determines productivity improvement. One of the reasons can be the plant intrinsic un-efficiency in using energy. Generally in full light only the 5 – 10% of the total incoming energy is allocated to net photosynthesis. Therefore preserving or improving this efficiency becomes pivotal for scientist and fruit growers. Even tough a conspicuous energy amount is reflected or transmitted, plants can not avoid to absorb photons in excess. The chlorophyll over-excitation promotes the reactive species production increasing the photoinhibition risks. The dangerous consequences of photoinhibition forced plants to evolve a complex and multilevel machine able to dissipate the energy excess quenching heat (Non Photochemical Quenching), moving electrons (water-water cycle , cyclic transport around PSI, glutathione-ascorbate cycle and photorespiration) and scavenging the generated reactive species. The price plants must pay for this equipment is the use of CO2 and reducing power with a consequent decrease of the photosynthetic efficiency, both because some photons are not used for carboxylation and an effective CO2 and reducing power loss occurs. Net photosynthesis increases with light until the saturation point, additional PPFD doesn’t improve carboxylation but it rises the efficiency of the alternative pathways in energy dissipation but also ROS production and photoinhibition risks. The wide photo-protective apparatus, although is not able to cope with the excessive incoming energy, therefore photodamage occurs. Each event increasing the photon pressure and/or decreasing the efficiency of the described photo-protective mechanisms (i.e. thermal stress, water and nutritional deficiency) can emphasize the photoinhibition. Likely in nature a small amount of not damaged photosystems is found because of the effective, efficient and energy consuming recovery system. Since the damaged PSII is quickly repaired with energy expense, it would be interesting to investigate how much PSII recovery costs to plant productivity. This PhD. dissertation purposes to improve the knowledge about the several strategies accomplished for managing the incoming energy and the light excess implication on photo-damage in peach. The thesis is organized in three scientific units. In the first section a new rapid, non-intrusive, whole tissue and universal technique for functional PSII determination was implemented and validated on different kinds of plants as C3 and C4 species, woody and herbaceous plants, wild type and Chlorophyll b-less mutant and monocot and dicot plants. In the second unit, using a “singular” experimental orchard named “Asymmetric orchard”, the relation between light environment and photosynthetic performance, water use and photoinhibition was investigated in peach at whole plant level, furthermore the effect of photon pressure variation on energy management was considered on single leaf. In the third section the quenching analysis method suggested by Kornyeyev and Hendrickson (2007) was validate on peach. Afterwards it was applied in the field where the influence of moderate light and water reduction on peach photosynthetic performances, water requirements, energy management and photoinhibition was studied. Using solar energy as fuel for life plant is intrinsically suicidal since the high constant photodamage risk. This dissertation would try to highlight the complex relation existing between plant, in particular peach, and light analysing the principal strategies plants developed to manage the incoming light for deriving the maximal benefits as possible minimizing the risks. In the first instance the new method proposed for functional PSII determination based on P700 redox kinetics seems to be a valid, non intrusive, universal and field-applicable technique, even because it is able to measure in deep the whole leaf tissue rather than the first leaf layers as fluorescence. Fluorescence Fv/Fm parameter gives a good estimate of functional PSII but only when data obtained by ad-axial and ab-axial leaf surface are averaged. In addition to this method the energy quenching analysis proposed by Kornyeyev and Hendrickson (2007), combined with the photosynthesis model proposed by von Caemmerer (2000) is a forceful tool to analyse and study, even in the field, the relation between plant and environmental factors such as water, temperature but first of all light. “Asymmetric” training system is a good way to study light energy, photosynthetic performance and water use relations in the field. At whole plant level net carboxylation increases with PPFD reaching a saturating point. Light excess rather than improve photosynthesis may emphasize water and thermal stress leading to stomatal limitation. Furthermore too much light does not promote net carboxylation improvement but PSII damage, in fact in the most light exposed plants about 50-60% of the total PSII is inactivated. At single leaf level, net carboxylation increases till saturation point (1000 – 1200 μmolm-2s-1) and light excess is dissipated by non photochemical quenching and non net carboxylative transports. The latter follows a quite similar pattern of Pn/PPFD curve reaching the saturation point at almost the same photon flux density. At middle-low irradiance NPQ seems to be lumen pH limited because the incoming photon pressure is not enough to generate the optimum lumen pH for violaxanthin de-epoxidase (VDE) full activation. Peach leaves try to cope with the light excess increasing the non net carboxylative transports. While PPFD rises the xanthophyll cycle is more and more activated and the rate of non net carboxylative transports is reduced. Some of these alternative transports, such as the water-water cycle, the cyclic transport around the PSI and the glutathione-ascorbate cycle are able to generate additional H+ in lumen in order to support the VDE activation when light can be limiting. Moreover the alternative transports seems to be involved as an important dissipative way when high temperature and sub-optimal conductance emphasize the photoinhibition risks. In peach, a moderate water and light reduction does not determine net carboxylation decrease but, diminishing the incoming light and the environmental evapo-transpiration request, stomatal conductance decreases, improving water use efficiency. Therefore lowering light intensity till not limiting levels, water could be saved not compromising net photosynthesis. The quenching analysis is able to partition absorbed energy in the several utilization, photoprotection and photo-oxidation pathways. When recovery is permitted only few PSII remained un-repaired, although more net PSII damage is recorded in plants placed in full light. Even in this experiment, in over saturating light the main dissipation pathway is the non photochemical quenching; at middle-low irradiance it seems to be pH limited and other transports, such as photorespiration and alternative transports, are used to support photoprotection and to contribute for creating the optimal trans-thylakoidal ΔpH for violaxanthin de-epoxidase. These alternative pathways become the main quenching mechanisms at very low light environment. Another aspect pointed out by this study is the role of NPQ as dissipative pathway when conductance becomes severely limiting. The evidence that in nature a small amount of damaged PSII is seen indicates the presence of an effective and efficient recovery mechanism that masks the real photodamage occurring during the day. At single leaf level, when repair is not allowed leaves in full light are two fold more photoinhibited than the shaded ones. Therefore light in excess of the photosynthetic optima does not promote net carboxylation but increases water loss and PSII damage. The more is photoinhibition the more must be the photosystems to be repaired and consequently the energy and dry matter to allocate in this essential activity. Since above the saturation point net photosynthesis is constant while photoinhibition increases it would be interesting to investigate how photodamage costs in terms of tree productivity. An other aspect of pivotal importance to be further widened is the combined influence of light and other environmental parameters, like water status, temperature and nutrition on peach light, water and phtosyntate management.

Retrieval of trace gases vertical profile in the lower atmosphere combining. Differential Optical Absorption Spectroscopy with radiative transfer models

The motivation for the work presented in this thesis is to retrieve profile information for the atmospheric trace constituents nitrogen dioxide (NO2) and ozone (O3) in the lower troposphere from remote sensing measurements. The remote sensing technique used, referred to as Multiple AXis Differential Optical Absorption Spectroscopy (MAX-DOAS), is a recent technique that represents a significant advance on the well-established DOAS, especially for what it concerns the study of tropospheric trace consituents. NO2 is an important trace gas in the lower troposphere due to the fact that it is involved in the production of tropospheric ozone; ozone and nitrogen dioxide are key factors in determining the quality of air with consequences, for example, on human health and the growth of vegetation. To understand the NO2 and ozone chemistry in more detail not only the concentrations at ground but also the acquisition of the vertical distribution is necessary. In fact, the budget of nitrogen oxides and ozone in the atmosphere is determined both by local emissions and non-local chemical and dynamical processes (i.e. diffusion and transport at various scales) that greatly impact on their vertical and temporal distribution: thus a tool to resolve the vertical profile information is really important. Useful measurement techniques for atmospheric trace species should fulfill at least two main requirements. First, they must be sufficiently sensitive to detect the species under consideration at their ambient concentration levels. Second, they must be specific, which means that the results of the measurement of a particular species must be neither positively nor negatively influenced by any other trace species simultaneously present in the probed volume of air. Air monitoring by spectroscopic techniques has proven to be a very useful tool to fulfill these desirable requirements as well as a number of other important properties. During the last decades, many such instruments have been developed which are based on the absorption properties of the constituents in various regions of the electromagnetic spectrum, ranging from the far infrared to the ultraviolet. Among them, Differential Optical Absorption Spectroscopy (DOAS) has played an important role. DOAS is an established remote sensing technique for atmospheric trace gases probing, which identifies and quantifies the trace gases in the atmosphere taking advantage of their molecular absorption structures in the near UV and visible wavelengths of the electromagnetic spectrum (from 0.25 μm to 0.75 μm). Passive DOAS, in particular, can detect the presence of a trace gas in terms of its integrated concentration over the atmospheric path from the sun to the receiver (the so called slant column density). The receiver can be located at ground, as well as on board an aircraft or a satellite platform. Passive DOAS has, therefore, a flexible measurement configuration that allows multiple applications. The ability to properly interpret passive DOAS measurements of atmospheric constituents depends crucially on how well the optical path of light collected by the system is understood. This is because the final product of DOAS is the concentration of a particular species integrated along the path that radiation covers in the atmosphere. This path is not known a priori and can only be evaluated by Radiative Transfer Models (RTMs). These models are used to calculate the so called vertical column density of a given trace gas, which is obtained by dividing the measured slant column density to the so called air mass factor, which is used to quantify the enhancement of the light path length within the absorber layers. In the case of the standard DOAS set-up, in which radiation is collected along the vertical direction (zenith-sky DOAS), calculations of the air mass factor have been made using “simple” single scattering radiative transfer models. This configuration has its highest sensitivity in the stratosphere, in particular during twilight. This is the result of the large enhancement in stratospheric light path at dawn and dusk combined with a relatively short tropospheric path. In order to increase the sensitivity of the instrument towards tropospheric signals, measurements with the telescope pointing the horizon (offaxis DOAS) have to be performed. In this circumstances, the light path in the lower layers can become very long and necessitate the use of radiative transfer models including multiple scattering, the full treatment of atmospheric sphericity and refraction. In this thesis, a recent development in the well-established DOAS technique is described, referred to as Multiple AXis Differential Optical Absorption Spectroscopy (MAX-DOAS). The MAX-DOAS consists in the simultaneous use of several off-axis directions near the horizon: using this configuration, not only the sensitivity to tropospheric trace gases is greatly improved, but vertical profile information can also be retrieved by combining the simultaneous off-axis measurements with sophisticated RTM calculations and inversion techniques. In particular there is a need for a RTM which is capable of dealing with all the processes intervening along the light path, supporting all DOAS geometries used, and treating multiple scattering events with varying phase functions involved. To achieve these multiple goals a statistical approach based on the Monte Carlo technique should be used. A Monte Carlo RTM generates an ensemble of random photon paths between the light source and the detector, and uses these paths to reconstruct a remote sensing measurement. Within the present study, the Monte Carlo radiative transfer model PROMSAR (PROcessing of Multi-Scattered Atmospheric Radiation) has been developed and used to correctly interpret the slant column densities obtained from MAX-DOAS measurements. In order to derive the vertical concentration profile of a trace gas from its slant column measurement, the AMF is only one part in the quantitative retrieval process. One indispensable requirement is a robust approach to invert the measurements and obtain the unknown concentrations, the air mass factors being known. For this purpose, in the present thesis, we have used the Chahine relaxation method. Ground-based Multiple AXis DOAS, combined with appropriate radiative transfer models and inversion techniques, is a promising tool for atmospheric studies in the lower troposphere and boundary layer, including the retrieval of profile information with a good degree of vertical resolution. This thesis has presented an application of this powerful comprehensive tool for the study of a preserved natural Mediterranean area (the Castel Porziano Estate, located 20 km South-West of Rome) where pollution is transported from remote sources. Application of this tool in densely populated or industrial areas is beginning to look particularly fruitful and represents an important subject for future studies.

Advanced applications of X-ray Absorption Spectroscopy to the study of protein metal sites.

We present a study of the metal sites of different proteins through X-ray Absorption Fine Structure (XAFS) spectroscopy. First of all, the capabilities of XAFS analysis have been improved by ab initio simulation of the near-edge region of the spectra, and an original analysis method has been proposed. The method subsequently served ad a tool to treat diverse biophysical problems, like the inhibition of proton-translocating proteins by metal ions and the matrix effect exerted on photosynthetic proteins (the bacterial Reaction Center, RC) by strongly dehydrate sugar matrices. A time-resolved study of Fe site of RC with μs resolution has been as well attempted. Finally, a further step aimed to improve the reliability of XAFS analysis has been performed by calculating the dynamical parameters of the metal binding cluster by means of DFT methods, and the theoretical result obtained for MbCO has been successfully compared with experimental data.

Development of new tools and devices for CMB and foreground data analysis and future experiments

The discovery of the Cosmic Microwave Background (CMB) radiation in 1965 is one of the fundamental milestones supporting the Big Bang theory. The CMB is one of the most important source of information in cosmology. The excellent accuracy of the recent CMB data of WMAP and Planck satellites confirmed the validity of the standard cosmological model and set a new challenge for the data analysis processes and their interpretation. In this thesis we deal with several aspects and useful tools of the data analysis. We focus on their optimization in order to have a complete exploitation of the Planck data and contribute to the final published results. The issues investigated are: the change of coordinates of CMB maps using the HEALPix package, the problem of the aliasing effect in the generation of low resolution maps, the comparison of the Angular Power Spectrum (APS) extraction performances of the optimal QML method, implemented in the code called BolPol, and the pseudo-Cl method, implemented in Cromaster. The QML method has been then applied to the Planck data at large angular scales to extract the CMB APS. The same method has been applied also to analyze the TT parity and the Low Variance anomalies in the Planck maps, showing a consistent deviation from the standard cosmological model, the possible origins for this results have been discussed. The Cromaster code instead has been applied to the 408 MHz and 1.42 GHz surveys focusing on the analysis of the APS of selected regions of the synchrotron emission. The new generation of CMB experiments will be dedicated to polarization measurements, for which are necessary high accuracy devices for separating the polarizations. Here a new technology, called Photonic Crystals, is exploited to develop a new polarization splitter device and its performances are compared to the devices used nowadays.

Fast and accurate numerical solutions in some problems of particle and radiation transport: synthetic acceleration for the method of short characteristics, Doppler-broadened scattering kernel, remote sensing of the cryosphere

The aim of this work is to present various aspects of numerical simulation of particle and radiation transport for industrial and environmental protection applications, to enable the analysis of complex physical processes in a fast, reliable, and efficient way. In the first part we deal with speed-up of numerical simulation of neutron transport for nuclear reactor core analysis. The convergence properties of the source iteration scheme of the Method of Characteristics applied to be heterogeneous structured geometries has been enhanced by means of Boundary Projection Acceleration, enabling the study of 2D and 3D geometries with transport theory without spatial homogenization. The computational performances have been verified with the C5G7 2D and 3D benchmarks, showing a sensible reduction of iterations and CPU time. The second part is devoted to the study of temperature-dependent elastic scattering of neutrons for heavy isotopes near to the thermal zone. A numerical computation of the Doppler convolution of the elastic scattering kernel based on the gas model is presented, for a general energy dependent cross section and scattering law in the center of mass system. The range of integration has been optimized employing a numerical cutoff, allowing a faster numerical evaluation of the convolution integral. Legendre moments of the transfer kernel are subsequently obtained by direct quadrature and a numerical analysis of the convergence is presented. In the third part we focus our attention to remote sensing applications of radiative transfer employed to investigate the Earth's cryosphere. The photon transport equation is applied to simulate reflectivity of glaciers varying the age of the layer of snow or ice, its thickness, the presence or not other underlying layers, the degree of dust included in the snow, creating a framework able to decipher spectral signals collected by orbiting detectors.

Genus Bifidobacterium: taxonomy studies and gene expression analysis on folate pathway

Folates (vitamin B9) are essential water soluble vitamins, whose deficiency in humans may contribute to the onset of several diseases, such as anaemia, cancer, cardiovascular diseases, neurological problems as well as defects in embryonic development. Human and other mammals are unable to synthesize ex novo folate obtaining it from exogenous sources, via intestinal absorption. Recently the gut microbiota has been identified as an important source of folates and the selection and use of folate producing microorganisms represents an innovative strategy to increase human folate levels. The aim of this thesis was to gain a fundamental understanding of folate metabolism in Bifidobacterium adolescentis. The work was subdivided in three main phases, also aimed to solve different problems encountered working with Bifidobacterium strains. First, a new identification method (based on PCR-RFLP of hsp60 gene) was specifically developed to identify Bifidobacterium strains. Secondly, Bifidobacterium adolescentis biodiversity was explored in order to recognize representing strains of this species to be screened for their folate production ability. Results showed that this species is characterized by a wide variability and support the idea that a possible new taxonomic re-organization would be required. Finally B. adolescentis folate metabolism was studied using a double approach. A quantitative analysis of folate content was complemented by the examination of expression levels of genes involved in folate related pathways. For the normalization process, required to increase the robustness of the qRT-PCR analysis, an appropriate set of reference genes was tested using two different algorithms. Results demonstrate that B.adolescentis strains may represent an endogenous source of natural folate and they could be used to fortify fermented dairy products. This bio-fortification strategy presents many advantages for the consumer, providing native folate forms more bio-available, and not implicated in the discussed controversy concerning the safety of high intake of synthetic folic acid.

Semi-synthetic bile acids as novel drug candidate in liver diseases: physico-chemical characterization and HPLC-ES-MS/MS methods for their quali-quantitative analysis in different experimental animal models

The physico-chemical characterization, structure-pharmacokinetic and metabolism studies of new semi synthetic analogues of natural bile acids (BAs) drug candidates have been performed. Recent studies discovered a role of BAs as agonists of FXR and TGR5 receptor, thus opening new therapeutic target for the treatment of liver diseases or metabolic disorders. Up to twenty new semisynthetic analogues have been synthesized and studied in order to find promising novel drugs candidates. In order to define the BAs structure-activity relationship, their main physico-chemical properties (solubility, detergency, lipophilicity and affinity with serum albumin) have been measured with validated analytical methodologies. Their metabolism and biodistribution has been studied in “bile fistula rat”, model where each BA is acutely administered through duodenal and femoral infusion and bile collected at different time interval allowing to define the relationship between structure and intestinal absorption and hepatic uptake ,metabolism and systemic spill-over. One of the studied analogues, 6α-ethyl-3α7α-dihydroxy-5β-cholanic acid, analogue of CDCA (INT 747, Obeticholic Acid (OCA)), recently under approval for the treatment of cholestatic liver diseases, requires additional studies to ensure its safety and lack of toxicity when administered to patients with a strong liver impairment. For this purpose, CCl4 inhalation to rat causing hepatic decompensation (cirrhosis) animal model has been developed and used to define the difference of OCA biodistribution in respect to control animals trying to define whether peripheral tissues might be also exposed as a result of toxic plasma levels of OCA, evaluating also the endogenous BAs biodistribution. An accurate and sensitive HPLC-ES-MS/MS method is developed to identify and quantify all BAs in biological matrices (bile, plasma, urine, liver, kidney, intestinal content and tissue) for which a sample pretreatment have been optimized.